We propose combination of the rabbit cornea endothelial and epithelial cells with bioengineered scaffolds in cell culture well inserts. This technology could significantly reduce the use of rabbits for the Draize rabbit eye irritation assay. The barrier to commercialization is the lack of an effective method for storing, banking, of living bioengineered cornea-equivalents. We will combine cell expansion and scaffold technology developed by our collaborator, Professor Soker at the Wake Forest Institute for Regenerative Medicine, with patented ice-free cryopreservation strategies developed by scientists at our company, Tissue Testing Technologies LLC. Cryopreservation using ice-free vitrification has shown promise with several human cell- derived construct models, including EpiOcular. This work will be performed in three specific aims: In the first aim a bioengineered gel scaffold originally formulated for human cells will be assessed for compatibility with rabbit endothelial and epithelial cells and optimized in culture well inserts. The scaffold and porous, clear insert membrane represents the stroma of the cornea. In the second aim we will compare three experimental groups, 1) corneal epithelial constructs without endothelial cells, 2) constructs with just an endothelium, and 3) cornea constructs with an endothelium under and an epithelium on top of the scaffold. They will be compared using established positive and negative control treatments to determine whether the less complicated constructs are more or less predictive of irritation than a construct with both endothelial and epithelial layers. In parallel with aim two the third aim will assess our lead ice-free cryopreservation strategy for short-term, 1-30 days, storage. During these studies a panel of biological and physical assays will be performed to determine the feasibility of employing our constructs for irritation evaluation of new chemical formulations. The Phase II plan will consist of further optimization, testing against a larger panel of irritants, long-term shelf-life evaluation and head-to-head comparison with the in vivo Draize rabbit eye test that will be required for translation, market acceptance, of cryopreserved bioengineered rabbit cornea-equivalents to the test market. !
Due to the hazardous nature of many materials and substances ocular toxicity testing is required to evaluate the dangers associated with new products ranging from household, industrial, agricultural, military, cosmetics, toiletries, and pharmaceutical products. The Draize eye irritation test in rabbits is commonly used. In vitro cell-based assays are frequently used for cosmetics and other research but none of the models are derived from the rabbit. In an effort to further reduce animal use for ocular testing, we will develop a rabbit cornea cell-based construct modeled upon the structure of the rabbit cornea. Since most in vivo ocular studies are in the rabbit it is logical to anticipate that a rabbit cell-based construct will be more likely be predictive of irritating, toxic outcomes for compounds in the rabbit eye than alternative models based on other species and cell types. The results will be compared with existing rabbit test data. If the model turns out to provide predictive data relevant to humans it may help reduce the number of compounds that need to be tested in whole animal studies.
